1. Field of the Invention
The present invention relates generally to the fields of immunology and protein chemistry. More specifically, the present invention relates to a novel cell system and methods of preparing apoenzyme.
2. Description of the Related Art
The major amine-degrading enzymes in the central nervous system and peripheral tissues of mammals are monoamine oxidase A and B MAO A and B; amine: oxygen, oxidoreductase (deaminating, flavin-containing), EC 1.4.3.4!. These isozymes are integral proteins of the outer mitochondrial membrane (1) and can be distinguished by differences in substrate preference (2), inhibitory specificity (3), tissue and cell distribution (4-6), and immunological properties (7-9). Furthermore, comparison of their nucleotide and deduced amino acid sequences show that human MAO A and B are two distinct proteins encoded by different genes (10).
Oxidation of amines by MAO is coupled to the reduction of an obligatory cofactor, FAD, which is covalently linked to the enzyme. Five types of bonds are generally found in the covalent linkage of flavins to their respective apoproteins (11). These include a histidine residue which can be attached through its N-1 or N-3 atom to the 8.alpha.-methyl group of the isoalloxazine ring to form a tertiary amine; a cysteine residue which forms a thioether linkage with either the 8.alpha.-methyl group or the C-6 of the xylene ring of the flavin molecule; or a tyrosine residue can become linked to the 8.alpha.-methyl group to form an (O)-8.alpha.-flavin bond. In MAO A and B, the 8.alpha.-methyl group of FAD is bound covalently to cysteine through a thioether linkage in the pentapeptide SGGCY (12, 13). Comparison of this segment with the complete deduced amino acid sequences of MAO A and B indicated that FAD is covalently bound to Cys.sup.406 in MAO A and Cys.sup.397 in MAO B, respectively (10). In addition, site-directed mutagenesis studies of MAO B, where Cys.sup.397 was substituted with serine or histidine, showed that this cysteine residue is essential for catalytic activity (14, 15).
Although the amino acid sequences surrounding the FAD covalent attachment site in different flavoproteins bear little homology, a distinct non-covalent FAD binding site displays high sequence identity in many FAD-containing enzymes of diverse function (16, 17). This non-covalent FAD binding region is commonly referred to as the dinucleotide binding site or motif due to its interaction with the AMP moiety of FAD. This motif consists of a .beta..sub.1 sheet-.alpha. helix-.beta..sub.2 sheet beginning with a highly conserved Gly-X-Gly-X-X-Gly sequence between the first .beta.-sheet and the .alpha.-helix. The second .beta.-sheet usually ends with a glutamate residue in which the .gamma.-carboxylate group is thought to interact through a hydrogen bond with the 2'-hydroxyl group of ribose in the AMP moiety of FAD. In MAO A and B, this motif is located at the N-terminus of MAO A (residues 15-43) and MAO B (residues 6-34), and ends in Glu.sup.43 and Glu.sup.34, respectively. Site-directed mutagenesis studies, where Glu.sup.34 was replaced with aspartate, glutamine or alanine, resulted in near complete or total loss of catalytic activity in MAO B (18).
A fundamental process in the intracellular generation of functional flavoenzymes is the molecular mechanism which generates holoenzyme from apoenzyme and its cofactor. Following the discovery of the first known enzyme with covalently linked FAD (succinate dehydrogenase, 19), extensive research in many laboratories has been conducted to elucidate how FAD is coupled to its respective proteins. The precise steps involved remain unknown.
The prior art is deficient in the lack of effective means of manipulating the flavinylation of enzymes and the preparation of apoenzymes. The present invention fulfills this longstanding need and desire in the art.